Staphylococcus aureus is one of the leading causes of life-threatening bacterial infections in both hospitals and community settings. This bacterium can colonize and invade virtually all tissues in the human body. The tremendous success of S. aureus as a pathogen is in part due to the temporal expression of an arsenal of virulence factors, which is executed by a series of two component regulatory systems (TCS) and helix-turn-helix proteins. Genetic analyses have revealed that at the center of the regulation of virulence factors in S. aureus are the transcription factors repressor of toxins (Rot) and the S. aureus exoprotein expression two-component system (SaeRS-TCS). Experiments using a murine model of systemic infection have revealed that S. aureus strains lacking rot are hypervirulent, while strains lacking sae are fully attenuated, highlighting the importance of these regulators for S. aureus pathophysiology. Rot is hypothesized to coordinate the differential expression of virulence factors by repressing the promoters of genes that code for cytotoxins and proteases, and activating the promoters of genes that code for surface proteins. In contrast, the SaeRS-TCS is involved in the activation of the promoters that code for most secreted factors, including cytotoxins. Comparison of the Rot and SaeRS regulons in clinically relevant strains has revealed that a large portion of Rot-repressed genes require the SaeRS-TCS for their activation. The precise molecular mechanism by which Rot regulates gene expression in S. aureus is not understood. Similarly, the mechanism by which Rot interferes with the SaeRS-TCS to alter the expression of target genes remains to be elucidated. Additionally, studies examining transcriptional and translational regulation of rot have primarily been done in vitro and do not necessarily mimic the impact this transcription factor has on virulence factor regulation in vivo. Thus, the Specific Aims of this proposal seek to: (i) define the mechanism of Rot-mediated regulation of virulence factors, and (ii) elucidate the regulation of rot transcriptio and translation ex vivo and in vivo. To accomplish these Aims, a multi-disciplinary approach will be employed where molecular biology, biochemistry, and bacteriology techniques will be combined with in vivo and ex vivo infection models. Collectively, the work proposed in this application will shed light into how Rot regulates the expression of virulence factors required for S. aureus pathogenesis. Due to the current epidemic of antibiotic-resistant S. aureus strains, understanding the intricacies of how S. aureus regulates the expression of virulence factors is likely to provide the foundation for the development of new anti-Staphylococcal drugs based on targeting regulatory circuits that are required for the pathogenesis of this bacterium.